Fig. 5
In vivo and in vitro validation of potential function of identified TSGs. a A schematic diagram illustrated how core regulators and epigenetic regulatory elements (including H3K27ac-marked distal enhancers, promoters, and super-enhancers) regulated well-known TSGs. Bar plot in the right indicated that there were more active TSGs in GI core regulator target genes than GII core regulator target genes. The p-value of Fisher-exact test showed in the plot. b The strategy used for selecting TSG candidates. Three super-enhancer-associated GI core regulators co-regulated genes were selected as TSG candidates. c Gene expression level of CLU in GI-like, GII-like, and normal samples in TCGA-LUAD cohort. The p-value of t-test showed in the plot. ns, not significant. d Track plots revealed CLU gene expression was regulated by GI upregulated distal enhancers. The CLU gene expression in GI and GII samples was shown on the right. e Real-time PCR quantification and Western blot detection of CLU in CRL-5803 (upper panel) and PC9 (lower panel) cells with or without CLU ectopic expression. f Cell proliferation assay in CRL-5803 (upper panel) and PC9 (lower panel) cells with or without CLU overexpression. g Soft agar colony formation assay in CRL-5803 (upper panel) and PC9 (lower panel) cells with or without CLU overexpression. h Real-time PCR quantification and Western blot detection of CLU in CRL-5872 cells with or without CLU knockdown. i Cell proliferation assay in CRL-5872 cells with or without CLU knockdown. j Soft agar colony formation assay in CRL-5872 cells with or without CLU knockdown. k Representative photos of HE and IHC staining of CLU and Ki-67 in GI and GII samples. l Statistical analyses of CLU and Ki-67 IHC scores in group I and group II samples. Data were shown as mean with S.E.M. Statistical analyses was calculated by two-tailed, unpaired t-test. m The TF binding motifs on the enhancer region. Grey boxes indicate TFs; red arrows, predicted sgRNAs; and yellow arrows, PCR primers for testing knockout efficiency. n Sequencing of the PCR products by reverse (R) primers to validate dual gRNA knockout efficiency. o CLU expression of the indicated sgRNAs by real-time PCR quantification. p Cell proliferation analysis of the indicated sgRNAs